Electrophoretic separation of compounds

ABSTRACT

A device, method and kit for the electrophoretic separation and purification of charged and neutral compounds in an analyte solution. The device comprises a chamber ( 1 ), at least one wall of which is composed of a chemical buffering system ( 4 ). A potential difference is applied across the buffering system, resulting in the charged and neutral compounds being differentially separated by extraction of the charged compounds into the buffering system. The device also comprises means for collecting the separated compounds, preferably in ampholyte-free or buffer-free solution and optionally means for recycling the separated fractions.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the electrophoretic separation, purificationand recovery of compounds from solution.

BACKGROUND ART

Complex mixtures such as biological samples can contain up to 30,000different proteins which need to be separated and identified for furtheranalysis. In proteome analysis, high resolution separation of complexprotein mixtures requires the development of novel techniques whichminimize separation times, are easy to use, result in a high degree ofpurity and allow for further analysis of the compound(s) of interestextracted from the sample without unnecessary additional purificationsteps.

2D-gel electrophoresis is one technique which is capable of separatingsuch complex biological samples (Wilkins, M. R. et al., ProteomeResearch: New Frontiers in Functional Genomics; Springer, 1997). With2D-gel electrophoresis, proteins are separated first by an isoelectricfocusing (IEF) step according to their isoelectric point. Secondly,proteins are separated as a function of their molecular mass by apolyacrylamide gel electrophoresis (PAGE) step. The result is atwo-dimensional image in which each visible spot corresponds to aspecific protein. If further analysis of a protein is required, forexample, analysis of peptide composition or biological activity, thenthe protein has to be first extracted from the gel matrix before it canbe analysed with the appropriate method to obtain the desiredinformation.

Methods have been developed to extract proteins from a polyacrylamide2D-gel, one such method consisting of cutting the gel around the proteinspot and extracting it in a wet chemical step. With this technique thereis a high probability that the protein will be denatured, modified oreven lost during its retrieval. Another technique is electroblotting,which is very time-consuming.

Once a protein is extracted from the gel, the most powerful analyticaltechnique is mass spectrometry. Using this technique, it is not onlypossible to analyse the peptide composition of proteins, but also tocompare the obtained peptide map to other protein data compiled in databanks by several bioinformatical institutions. In mass spectroscopy(MS), the purity of a sample is critical. If a sample containsimpurities such as salt, this is not directly amenable to MS analysis.Such a sample would require desalting before MS analysis by means suchas a dialysis procedure. Another way of avoiding undesired compounds inthe sample is to use a direct laser desorption technique from the 2D-gel(Ogorzalek Loo R. R., et al., Analytical Chemistry, 1996, 68, 1910-1917)or an electroblotted 2D-gel (Eckerskom, C. et.al., Analytical Chemistry,1997, 69, 2888-2892; Strupat, K. et al. Analytical Chemistry, 1994, 66,464-470). All of these additional purification steps complicate theanalysis procedure and are time-consuming.

While separation of compounds in complex mixtures is possible with2D-gel electrophoresis, there persists the problem of the numerousimpurities which remain together with the compounds desired foranalysis, removal of which is laborious. The major problem with 2Dgel-electrophoresis is that the compound of interest is trapped within agel and must be extracted and further purified before it can beanalysed.

Another method of separating complex biological samples is byisoelectric separation for example by iso-electric focusing (IEF)(Righetti, P. G., J. Biochem Biophys Methods, 1988 16:99-108). IEF is atechnique of electrophoresis whereby compounds can be separated on thebasis of charge within a pH gradient. In general, there are two majortypes of iso-electric focusing systems: (i) free flowing bufferedsystems and (ii) immobilised buffered systems.

(i) Free Flowing Buffering Systems

All free flowing systems are based on the use of a buffer, usuallycarrier ampholytes or isoelectric buffers such as amino acids. Forexample, a continuous free flow device has been demonstrated by Soulet,N. et al. (Electrophoresis, 1998, 19, 1294-1299). In this device, a pHgradient is created in a flat chamber using carrier ampholytes and apotential gradient perpendicular to the carrier flow direction. Thesample is continuously injected and partitioned at the end of the devicein discrete fractions. Although the pH gradient was stable over severalhours, a complete separation of bovine serum albumin andalpha-lactalbumin could not be achieved. Some major drawbacks of thissystem are that it is not able to separate compounds which have close p1values, that it takes several hours for the separation to occur and thatit also uses carrier ampholytes which need to be removed before furtheranalysis of the desired compounds is possible.

In isoelectric split-flow thin (SPLITT) fractionation, no pH gradient isestablished, but the separation principle is based on the charge thatproteins exhibit depending on their isoelectric point (pI) in buffers ofdifferent pH. A potential is applied to a flow cell using adequateoutlet and/or inlet splitters to separate the protein fractions. Twocomponent protein mixtures have been successfully separated (Fuh, C. B.and Giddings, J. C., Separation Science and Technology, 1997, 32,2945-2967), but this system exhibits some drawbacks when complex proteinsamples have to be analysed and when the isoelectric points (PI) ofproteins are very close (pI differences less than 0.1 pH unit are notpossible to separate using this method).

Many recycling isoelectric systems are based on the physical separationof compartments with different pH by means of membranes or screens. Someof them have been reviewed in the literature (Bier, M. Electrophoresis,1998,19,1057-1063; Krivankova, L. et al., Electrophoresis, 1998, 19,1064-1074). One of the most common preparative approaches to recyclingfree-flow electrophoresis is the Rotofor apparatus, commercialised byBioRad. In a tube-like apparatus where compartments are defined by ascreening material, the pH gradient is established using specialampholytes, the so-called Rotolytes. Gravity problems in free flowelectrophoresis are overcome by the rotation of the separationcompartments. This device has been successfully applied to thepreparative scale. A modification of this approach is the tangentialelectrophoretic apparatus from Bier, (U.S. Pat. No. 5,540,826). Here,the different compartments are arranged in such a manner that an arrayof multi-channels is separated from a second array of multi-channelsslightly displaced through a single screen. An electrical field isapplied perpendicularly to the channels which enables an electrophoreticserpentine pathway through the channels. The pH in the channels is fixedby ampholytes and recycling is possible with independent inlet andoutlet ports at every channel. The major disadvantages of this systemare that the device has a complicated construction of multi-channelsthrough which the solution must flow and that the compound(s) ofinterest remain(s) in an ampholyte solution which needs to be removedbefore further analysis of the desired compound or compounds ispossible.

In most solution-buffered systems, the analyte is mixed with a runningbuffer and several strategies of fluid handling are presented to eitherfractionate or desalt the sample or to work in a non convective and/orlow water diffusion medium. All these isoelectric focusing devices havea major disadvantage in terms of further analysis of compounds. They allcontain in the final separated fraction a certain amount of undesiredbuffering species or ampholytes.

(ii) Immobilized Buffering System

In most immobilized buffering systems, there is a major disadvantage interms of further analysis of compounds since the final separatedfraction is trapped in a gel or membrane.

There is a device developed by Righetti and Faupel (Righetti, P. G. etal. Journal of Chromatography, 1989, 475, 293-309) which is based on atechnique known as “segmented immobilized pH gradients”. The device iscomposed of multiple compartments sandwiched between an anodic and acathodic reservoir separated by immobiline isoelectric membranes,allowing the recovery of proteins in an ampholyte-free solution. Thisdevice can be composed of several compartments separated by immobilinegels stabilised by membranes. The separation of fractions is achieved insuch a way that the protein stops migrating in an electrical field inbetween two immobiline membranes, wherein one membrane establishes a pHhigher than the protein's pI and the other a pH lower than it. There areseveral disadvantages of this apparatus: the use of multiplecompartments, multiple immobilized membranes and segmented pH gradients.

SUMMARY OF THE INVENTION

The invention seeks to provide a device, method and kit for separatingcharged and neutral compounds and for the recovery of said neutralcompounds in a solution which can be an ampholyte-free or a buffer-freesolution. There exists a need in the art for a device, method and kitfor separation of compounds in complex mixtures combined with anefficient method of recovery of compounds of interest in order that theymay be further analysed, for example by mass spectrometry, withoutadditional time-consuming steps of extraction and purification whichexist in the prior art. The invention described herein can be used toseparate biological or chemical compounds within complex mixtures.

In one aspect, the present invention provides a device forelectrophoretic separation and purification of charged and neutralcompounds in an analyte solution, said device comprising: (a) a chamber,at least one portion of a wall of the chamber being composed of achemical buffering system; (b) a means for producing an potentialdifference across said chemical buffering system whereby said chargedand neutral compounds may be differentially separated by extraction ofsaid charged compounds into said chemical buffering system; (c) a meansfor collecting separated fractions, if desired in solution such as anampholyte-free or buffer-free solution and (d) optionally, means torecycle separated fractions.

In another aspect, the present invention provides a method ofelectrophoretic separation and purification of charged and neutralcompounds in an analyte solution and collection of separated fractionsusing the device of the present invention. A further advantage of thepresent invention is that compounds of interest are recoverable insolution, even in ampholyte-free or buffer-free solution.

The present invention relates to a method of separation and purificationwhich is fundamentally different from the prior art electrophoresistechniques, since the potential difference is not applied in the analytesolution nor between the analyte solution and the chemical bufferingsystem. In the present invention, the potential difference is onlyapplied through the chemical buffering system (or a portion thereof), insuch a manner that a portion of the electric field penetrates thechamber containing the solution to purify. This novel method andapparatus thereof has the advantage over prior art methods to increasethe resolution of the separation, to accelerate the purification speeddue to the migration in solution instead of in a gel or a membrane andto allow for direct fractionation for further analysis. It is notnecessary to use additional purification steps used in other prior artelectrophoretic devices and methods which use carrier ampholytes orisoelectric buffers that are commonly used to create a pH gradient inthe analyte solution.

In the present invention, the chemical buffering system may beadvantageously controlled with respect to the pH in its portioncontacting the analyte solution. When the compound or compounds ofinterest are globally neutral at the controlled pH, one is able toseparate the desired compound or compounds of interest from the mixture.Upon application of an electric field through the chemical bufferingsystem, preferably perpendicular to the analyte solution, it is possibleto discriminate between charged compounds and compounds that areglobally neutral at this pH. Indeed, the neutral compounds in contactwith the buffering system are maintained in the analyte solution,whereas the charged compounds migrate into the chemical bufferingsystem. In this manner, compounds may be separated by pI by controllingthe pH of the chemical buffering system.

The present invention therefore permits the electrophoretic separationand purification of compounds that are globally neutral from chargedspecies directly in an analyte solution which does not need to bebuffered. In some embodiments of this invention, the device has achamber having an inlet and outlet connected to a hydraulic flow system,wherein the analyte solution is capable of flowing through said chamber.In other embodiments, the device has a chamber in which the inlet andoutlet are merged, thereby constituting a simple reservoir in which themixture to purify can be deposited and from which the purified solutioncan be retrieved. In further embodiments, devices according to theinvention may contain a plurality of chambers for simultaneous and/orparallel purification. Preferably, the direction of the electricalcurrent is perpendicular to the direction of the flow of the analytesolution.

In preferred embodiments, the chemical buffering system has a defined pHvalue or a defined pH range, which may be achieved, for example, byusing covalently linked buffering molecules, amphoteric isoelectricmembranes, or any combination thereof. The chemical buffering system istherefore capable of separating the compound of interest by isoelectricpoint at a fixed pH or in a pH gradient. It is capable of separatingcompounds with different pI, for example compounds with differences inpI less than 0.1, compounds with differences in pI less than 0.01 andcompounds with differences in pI of up to 0.001, thereby permittingdifferent desired degrees of purification.

The chemical buffering system can be, for example, an immobiline gel, afluid solidified in a polymer matrix, a fritted glass, a porousmembrane, a filter or any combination thereof. This chemical bufferingsystem serves to control the pH in its portion contacting the analytesolution, thereby allowing discrimination between charged compounds andcompounds that are globally neutral at this pH. Said chemical bufferingsystem can thus be used to separate one or several neutral compounds ofinterest from a mixture containing charged compounds.

An electric current is applied across said chemical buffering system,and the shape of the chamber is designed in such a manner that theelectric current penetrates within this chamber, thereby generating amigration flux of the charged compounds present in solution. Thepurification efficiency and rate depend on the depth, on the width andlength or on the diameter of the chamber, and its geometrical shape canthus be chosen with respect to the purpose of the applications andexperiments to be carried out.

One advantage of the present invention is that the separation induced bythe migration of charged compounds allows the compound of interest to befractionated directly within the analyte solution. In this manner, theseparation is much faster than in prior art methods since the rate ofmigration is much faster in solution than in other kinds of mediacommonly used in the prior art such as gels or porous membranes wherethe high resistance drastically decreases the migration speed.

In some cases, the migrating charged molecules can penetrate into thechemical buffering system and further migrate within it. However, thismigration does not affect the separation within the analyte solution,and the chemical buffering system can be regarded as a waste reservoir.In some applications, it can be advantageous to prevent the adsorptionof the neutral compounds onto the wall of the chemical buffering system.In some embodiments of the present invention, means may be provided tostop direct absorption, comprising, for example, a fine membrane, whichcan be (for instance) a material of very low porosity.

In some instances, it can also be advantageous to recover the chargedmolecules that migrate within the chemical buffering system. To thisend, the device of the present invention may advantageously contain aplurality of sub-chambers.

In some embodiments of the present invention, an analyte solution can becaused to flow from an inlet an to outlet within the chamber. Computersimulation experiments show that the electric field applied through thechemical buffering system penetrates into the flow chamber, whichprovokes the migration of the charged species in the analyte solution(see examples). The computer simulation demonstrates that if theelectric field is directly applied between the extremities of thechamber containing the analyte solution, only a very small portion ofthe current lines penetrate into the chemical buffering system, forcingthe charged species to migrate within the analyte solution. This mode ofpolarisation has been used in prior art methods (as in segmented gelelectrophoresis), but to avoid these imperfections, the current withinthe chemical buffering system is increased by polarising the solution insuch a way that the ions are forced across the chemical buffering systemwhich is then used as a septum or a filter separating a network ofsolution compartments. In contrast to the prior art, the presentinvention embodies a separation principle fundamentally different sincethe potential difference is applied at both extremities of the chemicalbuffering system in such a manner that a portion of the electric fieldpenetrates the chamber containing the solution to purify.

The present invention has the advantage that a single chamber issufficient to perform the necessary separation, although a plurality ofchambers (or sub-chambers) can be used, if desired, in order to performsimultaneous separations at various desired pHs or parallel separationsat the same desired pH in the chemical buffering system. Furthermore,there is no limitation to the number, dimensions or shape of thechambers, which can be adapted to the specific required applicationwithout restriction of size, volume or quantity. Therefore, the devicesand methods of the invention can readily be scaled up or down. Forexample, it is possible to operate an analysis level, as well as atpreparative and pilot scales or in down stream processes.

In some embodiments, the devices of the present invention may have meansto control the temperature of the device and the analyte solution,particularly where high potential differences are employed.

In some preferred embodiments, devices according to the presentinvention may be provided with means to prevent precipitation of theneutral compound(s) of interest. Upon purification, the analyte solutionbecomes impoverished in ions, thereby decreasing its solubility. In suchcircumstances, the use of non-aqueous analyte solution or theintegration of a sonicator in the device of the present invention may beadvantageous.

In operation of devices according to the present invention, allcompounds that are charged at the pH established by the chemicalbuffering system migrate towards the extremities of the chamber,following the lines of current which depend on the position of theelectrodes, on the geometry of the whole device and on the nature ofboth the analyte solution and the chemical buffering system. In theanalyte solution remain only the compounds that are neutral at the pHdefined by the chemical buffering system.

The present invention permits rapid elimination of all undesired ions,including salts, charged acids or bases, buffer components or ampholytesfrom a solution. For instance, proteins can be purified in free flowingsolution and simultaneously prepared for further analysis. Similarly,the present invention can be used to isolate a neutral compound fromexcess charged by-products or salts.

In addition to the isolation of neutral species, the devices and methodsof the present invention also facilitate the extraction of chargedspecies, which, in operation, are loaded into the chemical bufferingsystem. For example, one important issue in proteome analysis is to haveaccess to low abundant proteins, the identification of which is oftenhampered by the presence of highly concentrated ones. For instance, thepresence of albumin in many cellular extracts prevents the detection ofproteins which are present in low concentration. In such applications,the present invention can for instance be used to separate albumin fromthe rest of the analyte solution by loading in an immobilised pHgradient (IPG) gel. To this end, the portion of the chemical bufferingsystem in contact with the analyte solution must have a pH rangeencompassing that of albumin or of any other compound that needs to beseparated from the rest of the analyte solution. In this manner, all thecompounds of the analyte solution that are charged in this pH range areextracted upon application of the electrical field into the IPG gel andare liable to migrate within this gel as long as they remain charged orup to the point where the pH of the gel corresponds to their respectivepI. In such cases, the compounds of interest are not only the neutralcompound(s) remaining in the analyte solution after electrophoreticpurification, but also—and sometimes mainly—the charged compounds thathave been extracted and that have migrated within the chemical bufferingsystem, since the latter are amenable to better determination andidentification than in the prior art.

In one mode of use, the present invention can be used to accumulatecompounds in the chemical buffering system. In such applications, theanalyte solution is renewed in the chamber, in such a manner that freshsolution is submitted to electrophoretic separation and purification. Inthis manner, compounds of low concentration in the analyte solution canbe accumulated in the chemical buffering system, thereby facilitatingtheir detection and identification. For identification purposes, it canalso be advantageous that the chemical buffering system be associatedwith means to specifically identify a compound or a class of compounds.Such means may operate to detect a compound or a class of compounds byfor instance emission of light, absorption of light (as in blotting),generation of an electroactive product, specific molecular recognition(for example as in the formation of an antigen-antibody complex or in anenzymatic reaction) that generates a detectable product.

In some embodiments, the device may be modified to facilitate therecovery of charged compounds that have been extracted from the analytesolution into the chemical buffering system. To this aim, the chambermay be divided into subchambers, at least one of them containing theanalyte solution. The other subchambers may be used for collecting thecompounds that migrate within the chemical buffering system andpreferably contain a buffer solution to fix the pH. As the chargedcompounds migrate along the direction of the electrical field and as aportion of this electrical field penetrates into each subchamber thecharged compounds can be extracted back from the chemical bufferingsystem into the subchambers. This migration continues until themigrating compounds reach a pH region of the chemical buffering systemor a subchamber where they are globally neutral. Such a configurationillustrates another advantage of the present invention, namely that itpermits the recovery of any compound in solution, even after migrationwithin the chemical buffering system. This is of great advantage withrespect to the prior art, because it greatly facilitates furtheranalysis of such recovered compounds.

The devices and methods according to the present invention have at leastthe following major advantages: (i) high sample recovery of compounds ofinterest directly in solution (ii) a high resolving power depending onthe pH interval across the isoelectric point (pI) of the desiredcompound (iii) a rapid separation rate due to the charged moleculesmigrating in solution rather than through denser materials such as gelsand (iv) separation of the desired compound of interest occurs directlyinto a ampholyte-free or buffer-free solution which convenientlyfacilitates further analysis without the need for extensive additionalpurification steps, such as desalting.

The compound or compounds of interest are preferably biologicalcompounds, more preferably organic compounds, and most preferably,proteins, protein derivatives, protein isoforms, enzymes, antigenes,antibodies, peptides or nucleic acids, lipids or carbohydrates.

In a yet further aspect, the invention provides a kit comprising thedevice of the present invention with instructions for theelectrophoretic separation and purification of charged and neutralcompounds in an analyte solution and, optionally, with the chemicals tomix or to use with the analyte solution to improve the purification ofthe desired compound(s). Such a kit permits the compound or compounds ofinterest to be recoverable in solution.

The compound of interest may be any biological or chemical compoundwhich is neutral at the pH or in the pH interval defined by the chemicalbuffering system in contact with the analyte solution. Preferably, thecompound of interest is an ionisable biological compound such as aprotein, an enzyme, a peptide or a compound containing a peptide orprotein moiety such as a glycoprotein, but can also be a nucleic acid,complex lipid or complex carbohydrate. It can also be any of variousisoforms of a protein or an antibody such as a monoclonal antibody.

The charged compound may be any compound that is charged at the pH or inthe pH interval defined by the chemical buffering system in contact withthe analyte solution. The charged compound can thus be either aionisable or charged compound, preferably an acid, a base, an ampholyteor a permanently charged compound like for example a dissociated salt.The charged compound is extracted from the chamber into the chemicalbuffering system upon electrophoretic separation according to thepresent invention. It can yet be further extracted out of the gel into asolution and may the compound of interest for the experimenter.

The analyte solution may be any solution according to the presentinvention which solubilizes the desired compound(s) of interest. It ispreferably an ampholyte-free or buffer-free solution.

The electric current can be applied by means of electrodes generated byan external power supply. Any voltage the device of the presentinvention can tolerate may be used (e.g. 10 to 10000 volts, preferably100 to 5000 volts). Higher voltages may be used provided that thegenerated heat can be dissipated by proper cooling. Additionally, thevoltage can be programmed to enable the application of any voltagewaveform, including alternative current and square wave.

The chamber is not limited in the number of subchambers, dimensions orshapes, which can be varied as required by the specific application ofthe device. The chamber may also be used as a module in conjunction withother separation, purification or detection components. The partscomprising the chamber may be machined from solid plastic such asplexiglass, moulded out of a thermoplastic resin, or made by any othersuitable manufacturing process. The material should have chemicalresistance to the analyte solution, the electric current, weak acids andbases, oxidants and so forth. In addition, it may be desirable to haveoptical clarity or at least some degree of transparency. The chamber maybe supported by an electrical insulating substrate made by an electricalinsulating material such as a porous membrane, a porous mineral layer, anon-conductive polymer (such as for example plexiglas) or a network ofelectrical insulating fibres. The chamber may also have a means toensure proper fit and positioning of the components in the chamber suchas, for example, the use of an O-ring to ensure that the chamber is flowtight to prevent leakage of the analyte solution, or, for example,screws to fasten the walls of the device together. Furthermore, thechamber is made of any material able to support voltages, like forexample plexiglass.

The chemical buffering system may be any system by which the separationof charged compounds from the compound(s) of interest can befacilitated. For example, the chemical buffering system can be a gel,most preferably an immobiline gel, a fluid solidified in a polymermatrix, a fritted glass, a porous membrane, a filter or any combinationthereof. The chemical buffering system can also have a means to stopdirect penetration of charged and neutral compounds in said chamber suchas low adsorption material. The chemical buffering system is mostpreferably thin, free of electroosmosis and flow-tight.

The pH can be a fixed pH or a pH gradient. For example, it can beproduced using covalently linked buffering molecules, such as forexample thiomorpholine derivatives or acrylamide derivatives. Thegradients of pH can be made by amphoteric isoelectric immobilizedpH-membranes, said membranes may have very short pH-gradients coveringonly a very narrow pH-interval. Ideally, said pH-interval can reach theultimate pH of the compound of interest to be separated and purified.With the method of the present invention, it may be possible to separatecompounds with pI difference up to a maximum resolution of pI to 0.001.

The hydraulic flow may be generated by means such as pressure,aspiration, centrifugal forces or electrical means. The direction of thehydraulic flow is at any suitable angle, most preferably perpendicular,with respect to the direction of the electrical current. The flow can bein single pass or recycled by means of fluid recycling loops.Furthermore, the flow can be distributed in a plurality of chambers, oneextremity of which can be interconnected to a single inlet or outlet.Other components may be included in the external flow channels, such asheat exchangers, reservoirs extending the volume capacity of eachrecirculating loop, and sensors such as for example a pH sensor atemperature sensor and/or a light absorption sensor.

The invention is hereinafter described in more detail by way of exampleonly, with reference to the attached figures which are briefly describedbelow.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Schematic representation of a separation device illustrating thepurification concept. The chamber 1 containing the solution to purifyhas one inlet 2 and one outlet 3, located at opposite extremities and itis covered by the chemical buffering system 4. The cathode 5 and theanode 6 are placed parallel to the chamber and are only in contact withthe chemical buffering system. The black arrows indicate the penetrationof positively (versus cathode) or negatively (versus anode) chargedcompounds into the chemical buffering system, whereas the white arrowsindicate that a flow of solution can be induced in the chamber.

FIG. 2. Simulation results for an electric field applied to a gel matrixand its effects on the solution for two values of the gel and solutionconductivity σ.

-   A) I: Potential distribution in the device when σ(gel)=σ(solution)    -   II: Current vectors when σ(gel)=σ(solution)-   B) III: Potential distribution in the device when    10σ(gel)=σ(solution)    -   IV: Current vectors when 10σ(gel)=σ(solution)

FIG. 3. Photograph of a prototype of separation device showing anarrangement similar to that represented in FIG. 1. The chamber 1possesses one inlet 2 and one outlet 3 that are connected to tubings topermit analyte solution flow through the device. The chemical bufferingsystem 4 is an immobilised pH gradient (IPG) gel placed above thechamber. The entire device is held in a screwed Plexiglas support 8, andits integrity is ensured by an o-ring 7 which allows a tight seal. Thecathode 5 and the anode 6 are placed in contact only with the IPG gel,close to the o-ring. These electrodes are made of a thin platinum wires,so that they can go above the o-ring without generating any leakage inthe device. When the gel reswells in the device, it encloses theelectrodes completely and prevents the analyte solution from touchingthe electrodes.

FIG. 4. A) Photograph of an immobiline gel at pH=7±0.14 pH units afterpurification in the separation device of FIG. 3 of a solution of IEFmarkers in water upon application of an electrical field of 100 V during1 hour. The migration of the negatively charged protein phycocyanin(band 9) to the anode and of the positively charged proteins likecytochrome c, myoglobin and haemoglobin (band 10) to the cathode isvisible by eye in this experiment. B) schematic diagram showing theseparation process in the immobilised pH gradient gel. The arrowindicates the direction of the pH gradient (low pH value at the anodicside of the gel and high pH value at the cathodic side of the gel).During the separation, a compound X with a pI corresponding to the pH ofthe portion of the gel in contact with the analyte solution is globallyneutral and does not migrate. A compound Y with a pI(Y)>pI((X) isnegatively charged in this pH range and migrates toward the anode,whereas a compound Z with pI(Z)<pI(X) is positively charged in this pHrange and migrates toward the cathode. The dotted arrows indicate thedirection of the migration of these various compounds.

FIG. 5. Purification experiment using a segment from pH 4-5.5 from animmobiline DryPlate.

-   A) Electropherogram obtained from CIEF analysis of IEF standards as    applied for the experiment. Peaks 11 to 21 corresponds to the    proteins of Table 1 below-   C) Electropherogram of the solution obtained after the experiment,    showing that only peaks 11 and 12 remain of large intensity after    purification.

FIG. 6. Purification of a mixture containing 5 different proteins,namely: trypsin inhibitor (pI=4.6, peak number 25), β-lactoglobuline B(pI=5.2, peak number 26), β-lactoglobuline A (pI=5.3, peak number 27),equine myoglobine (pI=7.0, peak number 28) and equine cytochrome c(pI=9.6, peak number 29).

-   A) CIEF analysis of: I) the applied protein mixture to purify; II)    the protein solution after the separation experiment with an    immobiline section of a pH range between 5.06 to 5.34.-   B) Scheme for the separation principle of the present experiment in    a separation device when using an immobiline gel with a pH gradient.    The protein of interest (noted A in the scheme) has a pI in between    the extremes of the pH gradient, i.e. between pH 5 and 5.4 in the    present case. Proteins symbolised by B and C in the scheme have a pI    larger than 5.4 or, respectively, smaller than 5.0, so that they are    positively and, respectively, negatively charged in the immobilised    pH gradient gel. The dotted arrows show the direction of the    migration of these various proteins, whereas the solid arrow shows    the direction of the pH gradient between the anodic and the cathodic    extremities of the gel (noted by a positive and, respectively a    negative sign). Upon application of the electric field, proteins of    types B and C migrate into the immobiline gel and are thus separated    from the proteins of type A.

FIG. 7. Mass spectrum (on the right side of the figure) obtained bysingle syringe injection of 2 μL of a solution of 80 μM catechine and 20μM methylene blue, including (on the left side of the figure) theevolution with time of the relative abundance of the peaks of mass290.5-291.5 (upper graph), of the peak of mass 285.5-286.5 (middlegraph) and of the total abundance of these two peaks (bottom graph).

FIG. 8. Mass spectrum (on the right side of the figure) obtained bycontinuous injection of a purified solution of 80 μM catechine and 20 μMmethylene blue, including (on the left side of the figure) the evolutionwith time of the relative abundance of the peaks of mass 290.5-291.5(upper graph), of the peak of mass 285.5-286.5 (middle graph) and of thetotal abundance of these two peaks (bottom graph). These results areobtained by on-line detection of the analyte solution that haspreviously flowed (without recycling) throughout the chamber of theelectrophoretic separation device (chemical buffering system: IPG gelwith the pH range 6-6.15 in contact with the analyte solution; appliedelectrical potential: 300 V, pumping rate 1 mL/min)

FIG. 9. Schematic representation of the set-up used for theelectrophoretic separation in a static mode in a device where the inletand outlet ends are merged, so that the chamber 31 is used as areservoir in which the analyte solution can be introduced beforepurification and retrieved after purification. The analyte solution isonly in contact with the chemical buffering system 32, and theelectrical potential is applied through the anode 33 and the cathode 34that are introduced in two reservoirs 35 and 36. The black arrowindicates the direction of the pH gradient introduced in the chemicalbuffering system.

FIG. 10. Photograph of an IPG gel after purification of a 300 mMmethylene blue and 10 mM phenol red water solution using a devicesimilar to that of FIG. 9 and subsequent determination of the migratedmethylene blue (spot number 39) and phenol red (spot number 38) to thecathodic and anodic reservoirs, respectively. The figure also shows thatno colour is present after electrophoretic separation in the portion 37of the gel that was in contact with the analyte solution during thepurification.

FIG. 11. Schematic representation of the set-up that can be used for theelectrophoretic separation in a static mode with on-line detection usingfor example an electrospray mass spectrometer. In this example, thedevice is supported in a plastic support 40 containing the chamber 41 incontact with the chemical buffering system 42. The chamber is made of aseries of three subchambers 40 in which the inlet and outlet ends aremerged, so that said subchambers are used as reservoirs in which theanalyte solution can be introduced before purification. Twosupplementary reservoirs 43 and 44 are used to introduce the electrodesserving to apply the electrical field necessary for performing theelectrophoretic purification. The subchambers also contain asupplementary connection system 45 (only one shown) for the coupling toanother apparatus 47 serving as supplementary separation step or as adetector. The figure shows that an electrical potential can be appliedbetween the subchambers (or a given position in the connection system)and the entrance of the apparatus 47 in order to control thehydrodynamic flow of the purified solutions and/or to generate anelectrospray 46, thereby allowing to detect the compounds of interestpresent in the purified analytical solution.

DETAILED DESCRIPTION EXAMPLE 1 Numerical Simulation of the Distributionof Migration Current within the Device of the Invention

In order to understand the distribution of the migration current in thedevice of the present invention, a numerical simulation can be run witha finite element calculation. Such experiments allow one to predict thecurrent flow through the purification device. To this end, FIG. 1 showsa schematic representation of an example of separation device andillustrates the purification concept. In this example, the deviceconsists of a chamber 1 containing the solution to purify with one inlet2 and one outlet 3 at each extremity of the chamber, of a chemicalbuffering system 4 in contact with a portion of the chamber and of twoelectrodes (a cathode 5 and an anode 6) that are only in contact withthe chemical buffering system and placed parallel to the chamber. Theblack arrows indicate the penetration of positively (versus cathode) ornegatively (versus anode) charged compounds into the chemical bufferingsystem upon application of an electrical field between the twoelectrodes, whereas the white arrows indicate that a flow of solutioncan be induced in the chamber.

A cross section of the device, composed of a chemical buffering systemas the cover of a channel is simulated, and the migration current iscalculated in each point of the section. Two different cases have forexample been simulated in the device where (i) the conductivity a isidentical in the gel and in the solution (σgel=a solution) and (ii) theconductivity in the gel is ten times lower than in the solution (10σgel=σsolution). For both cases, a calculation is solved in each pointof the structure following the Laplace equation:∇(−σ∇U)=0  (1)and using the appropriate conditions in a two-dimensional system:

-   at the first electrode: U=0,-   at the second electrode: U=1,-   at the insulating wall:

$\begin{matrix}{\left( \frac{\partial U}{\partial N} \right)_{wall} = 0} & (2)\end{matrix}$where U is the potential (V) and σ is the electrical conductivity (.⁻¹m⁻¹).

A stationary algorithm is used for the potential distribution. Thesimulations can be run using a commercial finite element software, FluxExpert® (Simulog, France) operating on a Unix workstation (SiliconGraphics Indigo 2 Solid Impact 10000 with 640 Mb RAM).

These simulation experiments aim at indicating whether the chargedcompounds migrate into the chemical buffering system or not, and atdemonstrating the influence of the conductivity σ on the migration or inother words, the effect of the buffer composition in the solution topurify. The obtained results are presented in FIG. 2.

In the first case (FIG. 2A), σ is considered to be equal in the chemicalbuffering system and in the analyte solution (σgel=σsolution). Apotential difference is applied between the two electrodes, which allowsthe prediction of the potential distribution. FIG. 2AI shows that thepotential distribution in the solution corresponding to the segmentunder the gel is closely similar to that in the chemical bufferingsystem. A potential gradient is also created in the chemical bufferingsystem, which can lead to a pre-migration of proteins in the solutiondepending on their charge. As shown in FIG. 2AII, the current vectorsindicate that the current is also transported through the solution. Thevectors are similar in the middle of the structure and lead to an equalcurrent flow. At the interface between the chemical buffering system andthe solution, it is clearly demonstrated that a current flow takes placefrom the solution to the chemical buffering system.

In the second case (FIG. 2B), the conductivity of the solution isenhanced. It is considered 10 times higher in the analyte solution thanin the gel (10 σgel=σsolution). The result of this experiment is thatthe potential gradient in the solution is less effective (see FIG.2BIII), but that more current is transported in the solution than in thechemical buffering system (see FIG. 2BIV). It can also be demonstrated,as in the first case, that a current flow takes place from the solutionto the chemical buffering system which enables the proteins to enter thechemical buffering system from the solution by migration.

From these two experiments, the concept of the separation andpurification device of the present invention may be demonstrated. Evenif the potential is only applied to the chemical buffering system, theanalyte solution adjacent to it is affected by this potential, and amigration of charged compounds (for example proteins) is induced. Thetwo cases differ only in their effectiveness. In the second case, ahigher conductivity is considered, corresponding to, for example, abuffered protein solution. This is certainly more desirable for theprotein stability and if the charge of some proteins have to bepre-selected for an isoelectric separation experiment. On the otherhand, it is clear that the first case favours protein migration andtherefore also the effectiveness of the purification device as nearly100% of the current is carried by proteins in, for example, anon-buffered solution (sample diluted in water).

EXAMPLE 2 Electrophoretic Separation and Purification in a Non-BufferedSolution

In order to demonstrate the electrophoretic separation and purificationof various solutions, the following experimental conditions have beenemployed:

Reagents

IEF protein marker standard is obtainable from BioRad (Herkules, US).Equine cytochrome c, B-lactoglobuline A and B, trypsin inhibitor andequine myoglobin can be purchased from Sigma. Immobiline DryPlates pHrange (4.5-5.4 and 4-7, 11 cm) are obtainable from Pharmacia Amersham.The reagents for capillary isoelectric focusing (CIEF) are allobtainable from BioRad.

Experimental Setup

A plastic holder can be constructed in such a manner that the solutionto purify can be pumped through the device containing the chambercontacting the chemical buffering system (which is an immobiline gel inthe present case). FIG. 2 shows a photograph of a prototype ofseparation and purification device that has such an arrangement. Thechamber 1 possesses one inlet 2 and one outlet 3 that are connected toteflon tubes and a peristaltic pump (not shown) in order to let theanalyte solution flow through the device. The chemical buffering system4 is an immobilised pH gradient (IPG) gel placed above the chamber. Theentire device is held in a screwed plastic support 8, and itswatertightness is ensured by an o-ring 7 which allows a tight seal. Thecathode 5 and the anode 6 are placed in contact only with the IPG gel,close to the o-ring. These electrodes are made of thin platinum wires,so that they can be integrated above the o-ring without generating anyleakage in the device. When the gel re-swells in the device, it enclosesthe electrodes completely and prevents the analyte solution fromtouching the electrodes. Re-swelling of the gels can be achieved for 1 hup to overnight in water or in the buffer system in which thepurification experiment can be carried out.

Purification

The different protein solutions (1 ml total volume) can be applied tothe device using the peristaltic pump. Before the experiment, thesolution can be circulated for at least 2 min, and a sample of 100 μlcan be taken for CIEF. A constant voltage varying from 30-100 Vaccording to the experiment can then be applied using a high voltagepower supply (Spellmann, CZE1000, New York, US). Voltage and current canbe recorded with a LabVIEW 5 program operated on a Digital PC and a dataacquisition board (Lab PC+, National Instruments, US).

Capillary Isoelectric Focusing (CIEF)

A Biofocus 3000 apparatus (BioRad, Hercules, US) can be used for CIEFanalysis using BioCap XL coated capillaries (ID 50 μm, BioRad). Theprotein samples can be diluted in ampholytes (Bio-Lyte, BioRad) andanalysed using BioRad IEF catholyte, anolyte and mobiliser. Whennecessary the samples can be ultracentrifugated with Biomax 5 kDafilters (Millipore, Bedford, Mass., US) prior to dilution in ampholytes,in order to guarantee sufficient concentration of proteins for the CIEFanalysis.

Photography

Digital photographs of the dried immobiline gels and the device afterthe experiments can be taken with a digital camera (Fuji MX-700, FujiPhoto Film, Tokio, Japan) and treated with Adobe Photoshop software.

EXAMPLE 2.1 Separation of Protein Markers at pH 7

In order to demonstrate protein migration as predicted in the simulationof the first case of Example 1 where the gel and solution have identicalconductivity, an immobiline gel of a pH range between 6.9-7.1 can beintegrated in a prototype of device as claimed by the present invention(see FIG. 3).

TABLE 1 Proteins in the IEF marker standard solution from BioRad andtheir corresponding pI and colour. Protein isoelectric point colourPhycocyanin 4.6 blue beta-lactoglobuline B 5.1 — bovine carbonicanhydrase 6.0 — human carbonic anhydrase 6.5 — equine myoglobin 7.0brown human haemoglobin A 7.1 red human haemoglobin C 7.5 red lentillectin III 7.8 — lentil lectin II 8.0 — lentil lectin I 8.2 — Cytochromec 9.6 brown

A solution of protein IEF markers in water (concentration of approx. 150mg/ml, protein composition see Table 1) can be applied and continuouslycirculated through the device of FIG. 3 using a peristaltic pump at aconstant pump rate (0.6 ml/min). A photograph of the immobiline gelafter 1 hour purification upon application of an electrical potential of100 V is shown in FIG. 4. For this experiment, the portion of the IPGgel in contact with the analyte solution has a pH of 7±0.14. This figureshows a blue band 9, indicating the migration of the blue colouredprotein phycocyanin towards the anode and a brown band 10, indicatingcytochrome c, myoglobin and haemoglobin migrating towards the cathode.

The proteins are concentrated in bands which demonstrate anelectrophoretic focalisation mechanism. This clearly indicates thatprotein migration is induced from the solution to the gel, although theelectrical potential is applied from electrodes only in contact with thegel. This also empirically confirms that the above simulation data agreewith the experiment.

EXAMPLE 2.2 Purification of beta-Lactoglobuline B and Phycocyanin from aIEF Marker Solution

In a further experiment, the purification of a solution consisting ofthe IEF marker proteins of Table 1 is demonstrated with a gel of pHrange 4-5.5 with the pH gradient parallel to the Platinum electrodes. ACIEF analysis is carried out before and after the purificationexperiment (pump rate 0.6 ml/min, constant voltage=50 V). As presentedin FIG. 5, the comparison of the two electropherograms demonstrates thatthe proteins of the original analyte solution with pI values higher than5.5 migrated into the gel, whereas beta-lactoglobuline B and phycocyanin(peaks 11 and 12) are still contained in the solution afterelectrophoretic purification.

A simple comparison can also be carried out by eye. The solution beforethe experiment is green (colour of the complex solution of IEF markers)whereas the solution is blue after the purification (corresponding tothe colour of phycocyanin). Additionally, the gel only exhibits a browncolour at the cathode side, corresponding to the positively chargedproteins migrating towards it.

This experiment demonstrates that an analyte solution containingcompounds of interest can be purified by extraction of charged compoundsusing a device and method of the present invention.

EXAMPLE 2.3 Purification of beta-Laglobulines A and B

A protein solution consisting of five proteins with known isoelectricpoints (trypsin inhibitor (pI=4.6), beta-lactoglobulin A (pI=5.3),beta-lactoglobulin B (pI=5.2), equine myoglobin (pI=7.0), cytochrome c(pI=9.6), at a concentration of 200 μg/ml except trypsin inhibitor with50 μg/ml in water) is applied to the device of FIG. 3 containing animmobiline gel of a pH range from 5 to 5.4, rehydrated in water. Asillustrated in the scheme of the separation process of FIG. 6B, the aimof this experiment is to recover beta-lactoglobulines A and B insolution. To this aim, the purification is based on the followingprinciple: proteins with 5.0<pI<5.4 are either charged negatively in thegel near the cathode and repelled (pH in gel>pI), as illustrated by theproteins of type A in FIG. 6B. On the other more acidic gel extremitynear the anode, these proteins of type A are positively charged (pH ingel<pI) and again repelled. In this manner, they cannot be extractedfrom the analyte solution. All other proteins with pI>5.4 are positivelycharged and attracted to the cathode (proteins of type B in FIG. 6B),whereas all proteins with pI<5.0 are attracted by the anode (proteins oftype C in FIG. 6B). These last two types of compounds are thus extractedinto the IPG gel upon electrophoretic purification of the analytesolution.

The electropherograms of the solution of the five above proteins areexamined before and after purification and the results reported in FIG.6AI and II show that the proteins trypsin inhibitor, equine myoglobinand equine cytochrome c disappeared nearly totally after purification,whereas the two beta-lactoglobulins stay in the solution. This is aclear proof for the purification principle based on isoelectricseparation according to the present invention.

One advantage of the device as claimed in this invention is that theproteins to be purified are in minimal contact to the immobiline matrix,which reduces possible effect the polyacrylamide matrix could have onthe proteins. They can be recovered easily in solution for furtheranalysis. No extraction with chemicals needs to be carried out,minimising the effect of chemicals to the protein of interest. This factalso reduces the purification time. We could show here that thepurification of microgram quantities can be carried out in 1 h. It mayeven be enhanced with the use of a cooling device or a differentgeometry ensuring less current flow through the device. This would allowthe application of a higher electrical potential.

EXAMPLE 3 Electrophoretic Separation in a Buffered Solution

To test the simulation of the second case of the simulation experimentof example 1 where the conductivity of the gel is ten times lower thatthe solution, the solution of the protein markers of Table 1 is adjustedto a given pH. An acetate buffer (0.01 M) with a pH of 4.6 is used forthis purpose. This pH corresponds to the pI of phycocyanin which iscontained in the IEF marker standard (see Table 1). The pH range of thegel varied between 4.5-4.58 and 4.58-4.66. In these experiments, thecurrent is set constant to 300 μA, which is the upper limit of the powersupply. The voltage that has been detected to never exceed 30 V. Afterseveral hours of electrical potential application, only very littleprotein is visible in the gel (results not shown). These proteins arevery diffuse and not focused in a band as in the above experiments.Also, bubble formation is enhanced, thereby causing a certaindestruction of the gel in the device.

These experiments clearly show that the migration efficiency of theproteins is dramatically decreased if the sample solution is buffered.It is clear that more current is carried by buffering ions, when theirconcentration is high in comparison to that of the protein mixture. Onthe contrary, the current is mainly transported by the proteinsthemselves when they are contained in water only. This favours proteinmigration and therefore the separation efficiency of the device. Whilewater is not the most favoured analyte solvent for proteins, the abovemethod does not need any addition of buffer ions or ampholytes for theenhancement of isoelectric separation. In a practical point of view,this greatly facilitates the separation process.

EXAMPLE 4 Electrophoretic Purification Coupled with On-Line MassSpectrometry Detection

A device similar to that shown in FIG. 3 can be coupled to a massspectrometer (LCQ-DUO, Finnigan) for on-line detection of the compoundor compounds of interest. To this aim, a mixture of 80 μM catechine and20 μM methylene blue can be pumped through the electrophoreticseparation device at a rate of 1 mL/min (using a peristaltic pump fromIsmatec). The device contains a chemical buffering system made of an IPGgel of pH 5.5 to 6.5, so that the portion of the gel in contact with thechamber exhibits the pH range 6-6.15. The outlet end of the chamber isconnected by tubings to the injection system of a LCQ-DUO massspectrometer for on-line analysis of the solution.

Catechine is a well-known mass marker that is neutral between pH 6 and6.15, whereas methylene blue is a permanent cation. When this mixtureflows in the device of the present invention, methylene blue isextracted out of the analyte solution and penetrates into the IPG gelupon application of an electrical potential (for example 300 V). In thismanner, methylene blue is eliminated from the solution, and thecatechine is purified. This is evidenced in FIGS. 7 and 8 that show themass spectrogram of the analyte solution before and, respectively, afterelectrophoretic purification. To this end, the results of FIG. 7 havebeen obtained with 1 μL of the starting analyte solution that has beenelectrosprayed from a syringe in the mass spectrometer (in atmosphericpressure chemical ionisation (APCI) mode, with nitrogen as sheath gasand with the following working conditions: voltage source: 3.82 kV;current source: 5.4 mA; vaporizer temperature: 450° C.; sheath gas flowrate: 79.9 psi; capillary voltage: 4.6 V and capillary temperature: 200°C.). The obtained spectrum mainly shows two very strong peaks at themass/charge (m/z) ratios of 286.3 and 291 corresponding to methyleneblue and catechine, respectively. The intensity of the peak at m/z=291is only about 60% that of the peak at m/z=286.3, in agreement with thelarger concentration of methylene blue in the analyte solution. Afterelectrophoretic purification of the analyte solution, the mass spectrumof FIG. 8 exhibits a similar shape, but the relative abundance of thepeaks become almost the same (the intensity of the peak at m/z=291 is94% that of the peak at m/z=286.3). The experiment can be run further,and the evolution of the relative abundance of the two peaks with timeshows that the intensity of the peak at m/z=291 remains approximatelyconstant, whereas that of the peak at m/z=286.3 passes from 100% to lessthan 40% within less than two minutes.

These results clearly indicate that the analyte solution has beenpurified, in agreement with the blue band of methylene blue observed inthe gel close to the cathode. The length of the chamber (about 3 cm) isnot sufficient to completely eliminate methylene blue from the analytesolution, but the dimension of the chamber, the flow rate of theanalytical solution as well as the value of the electrical field can beoptimised to allow complete purification.

This experiment clearly demonstrates that the device of the presentinvention can be coupled to a mass spectrometer for on-line detection ofthe purified solution. In this manner, further separation or detectionof the purified solution can be easily conducted. In some applications,the purified fractions can also be collected in another support beforefurther analysis, like for instance a MALDI (matrix assisted laserdesorption ionisation) plate.

EXAMPLE 5 Electrophoretic Purification of Isoforms

In order to demonstrate the separation and purification of proteinisoforms, N-acetyl Eglin C is obtainable by recombinant DNA techniquescontaining two isoforms (one in basic pH and one in acid pH range).

A water solution of 1 mg/mL N-acetyl Eglin C can be recycled in thedevice of the present invention and run constantly at 1000 volts for 1hour on an immobilsed pH gradient gel at pH 5.5 (pI of N-acetyl EglinC).

The results that can be obtained using a conventional capillaryisoelectric focusing apparatus (Biofocus, Bio-Rad) show that the analytesolution to purify presents one peak after 26.86 min. (corresponding tothe basic isoform pI 6.2: 4.86%), a main peak after 29.56 min.(corresponding to Eglin C: 90.18%) and a peak after 31.52 min.(corresponding to the acid isoform pI 5.2: 4.94%). After separation andpurification according to the method of the present invention, thepurified solution shows a very small peak corresponding to a trace ofbasic isoform at 26.38 min. And a peak at 29.57 min. (97.88%)corresponding to the main component of N-acetyl Eglin C. No peakcorresponding to the acid isoform is present, demonstrating the isoformseparation and purification and the enrichment of the main component.

EXAMPLE 6 Electrophoretic Purification in Static Mode

For certain applications, it may be advantageous to purify the analytesolution without hydraulic flow. In such cases, the device of thepresent invention does not require a chamber with inlet and outlet ends,but only a reservoir to introduce the analyte solution and retrieve it.

This is exemplified in FIG. 9 which shows a schematic representation ofthe set-up used for the electrophoretic separation in a static mode in adevice where the inlet and outlet ends are merged, so that the chamber31 is used as a reservoir in which the analyte solution can beintroduced before purification and retrieved after purification. Theanalyte solution is only in contact with the chemical buffering system32, and the electrical potential is applied through the anode 33 and thecathode 34 that are introduced in two reservoirs 35 and 36. The blackarrow indicates the direction of the pH gradient introduced in thechemical buffering system.

For the demonstration of the separation with such a device of thepresent invention, one can fabricate an electrophoretic separationdevice similar to that shown in FIG. 9 which includes an immobilised pHgradient (IPG) gel serving as chemical buffering system and a chambercontaining three sub-chambers consisting of small plastic tubes that areplaced on the top of the IPG gel and disposed along the direction of thepH gradient. As schematically illustrated in FIG. 9, the analytesolution can be introduced into the central subchamber, whereas the twoother subchambers are filled with water and contain each an electrode soas to serve as cathodic and anodic reservoirs, respectively. In thismanner, the electrodes are not directly in contact with the analytesolution. The electrical field has to pass through the IPG gel, and aportion of the electric field penetrates into the subchamber containingthe analyte solution to purify.

To demonstrate the separation and purification of an analyte solutionwith such a configuration of electrophoretic device, an immobiline gel(pH range 4-7) can be reswelled in water overnight at room temperature.Three plastic wells (1 cm diameter) with holes (0.8 cm in diameter)opened in their bottom can be placed on top of the IPG gel, respectivelyon the pH 4.5, pH 5.5 and pH 6 lines. One hundred μL of a 300 μMmethylene blue and 10 mM phenol red water solution can then be depositedin the central well in contact with the gel at pH 6. Two platinumelectrodes can be respectively placed in the right and left side wellswhich are filled with water.

In these conditions, both compounds are charged over the whole pH rangeimposed by the IPG gel, since methylene blue is a permanent cation andphenol red is negatively charged below its pKa which has a value of7.81. Methylene blue exhibits a blue colour whereas phenol red is yellowin its anionic form, so that extraction of both analytes out of theanalyte subchamber into the IPG gel upon application of an electricalpotential can be easily identified. Indeed, upon application of aconstant voltage (500 V) between the two platinum electrodes using ahigh voltage power supply (Landis & Gyr), it can be seen that methyleneblue migrates towards the cathode, whereas phenol red migrates towardsthe anode. After one hour of purification, a digital photograph of thegel is taken with a numerical camera (Camedia C-2020 Z—Olympus) andtreated with Olympus Camedia software. This photograph of the IPG gelpresented in FIG. 10 shows that the purification is complete, which isdemonstrated by the fact that the central reservoir is colourless(absence of colour in the portion 37 of the gel that was in contact withthe analyte reservoir), whereas the portion of the gel below the anodicreservoir is yellow (spot 39 in FIG. 10) and that below the cathodicreservoir is blue (spot 39 in FIG. 10).

These results clearly demonstrate the efficiency of the method of thepresent invention, even when no flow is induced to the analyte solutionto purify. However, agitation may be induced either to the subchambersor to the entire device, so as to increase the convection. As theefficiency and rate of the separation depends on the migration of thecharged compound in the analyte solution, it may be advantageous toavoid the formation of concentration gradients and hence to insurehomogeneity of the analyte solution. For certain applications likeprotein purification, it can also be advantageous to control thetemperature of the subchambers and to add means of avoidingprecipitation (for example by sonicating the subchambers).

It is worth noting here that the solutions in the anodic and cathodicreservoirs can be slightly coloured at the end of the purification. Insuch a case, this indicates that part of the methylene blue and part ofthe phenol red are extracted out of the IPG gel into the anodic and,respectively, the cathodic reservoirs, thereby allowing to recover insolution the compounds that have been extracted from the analytesolution into the chemical buffering system. This can be useful in manyapplications and demonstrates one interest of disposing a plurality ofsub-chambers in the separation device so as to collect various purifiedfractions, as specified in some embodiments of the present invention.

An example of purification device containing a plurality of subchambersis shown in FIG. 11 which is a schematic representation of the set-upthat can be used for the electrophoretic separation in a static modewith on-line detection or connection to a further separation step. Inthis illustration, the device is supported in a plastic support 40containing the chamber 41 in contact with the chemical buffering system42. The chamber is made of a series of subchambers 41 in which the inletand outlet ends are merged, so that said subchamber are used asreservoirs in which the analyte solution can be introduced beforepurification and retrieved after purification. Only three subchambersare represented here, but there is no limitation in the number,disposition and shape of these subchambers. Two supplementary reservoirs43 and 44 are used to introduce the electrodes serving to apply theelectrical field necessary for performing the electrophoreticpurification. The subchambers also contain a supplementary connectionsystem 45 (only one shown) for the coupling to another apparatus 47serving as supplementary separation step or as a detector. The figureshows that an electrical potential can be applied between thesubchambers (or a given position in the connection system) and theentrance of the apparatus 47 in order to control the hydrodynamic flowof the purified solutions and/or to generate an electrospray 46, therebypermitting detection of the compounds of interest present in thepurified analytical solution.

Finally, recovery of the compound or compounds of interest in solutiongreatly facilitates further separation, purification and/or detection.To this end, the subchambers of the devices described in the presentexperiment can contain a connection (like for example an aperture, agroove, a sealed tube, a capillary, a sealed micro-channel or any othercoupling system) that allows on-line introduction or injection of thepurified solution into another detection system (see FIG. 11 for anexample). Such a system can be demonstrated with a conventional liquidchromatograph that is used for example to further separate a cellularextract that has been purified by the electrophoretic method of thepresent invention and that contains several compounds of interest thatneed to be identified individually. Similarly, the subchambers of thepresent device can for example be directly coupled to a massspectrometer (with direct sampling using aspiration, mechanical orelectrokinetic pumping), thereby allowing on-line identification of thecompound or compounds of interest.

All publications and patent applications cited in this specification areherein incorporated by reference. Although the foregoing invention hasbeen described in some detail by way of illustration and examples forclarity of understanding, it will be readily apparent to a personskilled in the art in light of the teachings of this invention thatcertain changes and modifications may be made thereto without departingfrom the spirit or scope of the appended claims.

1. A device for electrophoretic separation and purification of chargedand neutral compounds in an analyte solution, said device comprising:(a) a chamber containing said analyte solution, at least a part of onewall of the chamber being composed of the buffering face of a firstportion of a chemical buffering system, said chemical buffering systemdefining a desired pH or pH gradient in said first portion contactingsaid analyte solution, said first portion being integral with aremaining portion of said chemical buffering system extending outside ofsaid chamber, said first portion enabling the charged compounds tomigrate from said analyte solution in said chamber into said remainingportion of said chemical buffering system and out of said chamber whilethe neutral compound and said analyte solution remain within saidchamber; and (b) means for producing a potential difference across saidchemical buffering system to generate an electric field that isdistributed along said buffering face of said chemical buffering systemand that penetrates into said analyte solution in said chamber at aposition of said first portion of said chemical buffering system incontact with said analyte solution, whereby compounds that are globallyneutral at said pH or pH range defined by said first portion of saidchemical buffering system contacting said analyte solution remain insaid chamber upon potential application, whereas compounds that arecharged at said pH or pH range defined by said first portion of saidchemical buffering system contacting said analyte solution migrate andare extracted out of said chamber into said chemical buffering systemupon potential application.
 2. A device according to claim 1 whereinsaid chamber has an inlet and an outlet connected to a hydraulic flowsystem, wherein the analyte solution is capable of flowing through saidchamber.
 3. A device according to claim 1 wherein the chamberconstitutes a reservoir in which the analyte solution can be introducedbefore separation and purification and from which the purified solutioncan be retrieved.
 4. A device according to claim 1, wherein the desiredpH or the pH gradient in said chemical buffering system is producedusing covalently linked buffering molecules.
 5. A device according toclaim 1, wherein said chamber has an inlet and a plurality of outlets,thereby allowing the collection of fractions at different portions ofthe chemical buffering system.
 6. A device according to claim 1, whereinsaid chamber has a plurality of sub-chambers, said sub-chambers beinginterconnected by said chemical buffering system, thereby defining adesired pH value or a desired pH gradient in each sub-chamber.
 7. Adevice according to claim 1, wherein said means for producing saidpotential difference along said chemical buffering system compriseselectrodes.
 8. A device according to claim 1, wherein said chemicalbuffering system is capable of isoelectric separation of at least onecompound of interest at a defined pH value or in a defined pH range. 9.A device according to claim 1, wherein said chemical buffering systemdefines in said first portion contacting said analyte solution in saidchamber a pH gradient of less than 0.1 pH unit.
 10. A device accordingto claim 1, wherein said chemical buffering system contains means fordirect identification or quantification of one of a compound and a classof compounds that have been extracted from the analyte solution out ofthe chamber.
 11. A device according to claim 10, wherein saididentification and/or quantification means is based on the production oflight, the absorption of light, the reaction with a blotting agent orlabel, the generation of an electroactive species or the specificmolecular recognition of compounds.
 12. A device according to claim 1,further comprising a fine membrane separator that stops directadsorption of the neutral compounds onto the chemical buffering systemwall.
 13. A device according to claim 1, further comprising means tocontrol the temperature of said device and the analyte solution.
 14. Adevice according to claim 1, further comprising coupling meanspermitting purified analyte solution or recovered charged compounds tobe passed from the device into other separation or detection systems.15. A device according to claim 1, wherein said device is multiplexed toperform one of simultaneous and parallel electrophoretic separation andpurification of charged and neutral compounds.
 16. A device according toclaim 1, wherein said chemical buffering system is selected from a groupconsisting of a gel comprising immobilised buffering molecules and abuffering fluid solidified in one of a polymer matrix, a fritted glass,a porous membrane, a filter and a combination thereof.
 17. A deviceaccording to claim 1, further comprising means for collecting separatedfractions in an ampholyte-free or buffer-free solution.
 18. A deviceaccording to claim 1, further comprising means to recycle separatedfractions in said chamber.
 19. A device according to claim 1, furthercomprising means to pump said analyte solution in said chamber.
 20. Adevice according to claim 19, wherein a direction of the electricalfield is substantially perpendicular to a direction of a flow of theanalyte solution within the chamber.
 21. A device according to claim 1,wherein a length of said chamber is substantially perpendicular to adirection of the pH gradient of the chemical buffering system and to adirection of the electric field.
 22. A device according to claim 1,further comprising electrodes that are in contact with said chemicalbuffering system on opposite sides of said chamber a spaced distancefrom said chamber so as not to be in direct contact with the analytesolution.
 23. A device according to claim 1, wherein said means forproducing said potential difference along said chemical buffering systemcomprises electrodes integrated within the chemical buffering system.24. A method of electrophoretic separation and purification of chargedand neutral compounds in an analyte solution and collection of separatedfractions comprising the steps of: (a) providing a device comprising:(i) a chamber containing said analyte solution, at least a part of onewall of the chamber being composed of the buffering face of a firstportion of a chemical buffering system, said chemical buffering systemdefining a desired pH or pH gradient in the first portion contactingsaid analyte solution, the first portion being integral with a remainingportion of said chemical buffering system extending outside of thechamber, the first portion enabling the charged compounds to migratefrom said analyte solution in the chamber into the remaining portion ofsaid chemical buffering system and out of the chamber while the neutralcompound and said analyte solution remain within the chamber; and (ii)electrodes adapted to generate an electric field that is distributedalong said buffering face of said chemical buffering system and thatpenetrates into said analyte solution in the chamber at a position ofthe first portion of the chemical buffering system in contact with theanalyte solution, (b) applying a potential difference between saidelectrodes so that compounds that are globally neutral at the pH or pHrange defined by the first portion of the chemical buffering systemcontacting the analyte solution remain in the chamber upon potentialapplication, whereas compounds that are charged at the pH or pH rangedefined by the first portion of the chemical buffering system contactingthe analyte solution migrate and are extracted out of the chamber intosaid chemical buffering system.
 25. A method according to claim 24,wherein the compounds are biological compounds, organic compounds,proteins or protein derivatives, or isoforms.
 26. A method according toclaim 24, wherein the analyte solution is one of a non-aqueous solutionand a solution containing an organic solvent.
 27. A method according toclaim 24, wherein the device is used to load the chemical bufferingsystem with compounds of interest.
 28. A method according to claim 27,wherein the analyte solution is renewed in the chamber in order toaccumulate compounds of interest in the chemical buffering system.
 29. Amethod according to claim 24, further comprising the step of recyclingthe analyte solution and the separated fractions in said chamber.
 30. Amethod according to claim 24, further comprising the step of recoveringthe separated fractions in solution.
 31. A method according to claim 30,wherein the separated fractions are recoverable in one of anampholyte-free solution and a buffer-free solution.
 32. A methodaccording to claim 24, further comprising the step of pumping theanalyte solution through said chamber during the separation.
 33. Amethod according to claim 24, wherein a direction of the pH gradient ofthe chemical buffering system and a direction of the electric field issubstantially perpendicular to a length of the chamber.
 34. A kitcomprising a device and instructions for electrophoretic separation andpurification of charged and neutral compounds in an analyte solution,said device comprising a chamber for containing the analyte solution anda chemical buffering system arranged in such a manner that: (a) at leasta part of one wall of the chamber is composed of the buffering face of afirst portion of said chemical buffering system, said chemical bufferingsystem defining a desired pH or pH gradient in said first portion incontact with said analyte solution, said first portion being integralwith a remaining portion of said chemical buffering system extendingoutside of said chamber, said first portion enabling the chargedcompounds to migrate from said analyte solution into said remainingportion of said chemical buffering system and out of said chamber whilethe neutral compound and said analyte solution remain within saidchamber; (b) said device having means for producing a potentialdifference across said chemical buffering system to generate an electricfield that is distributed along said buffering face of said chemicalbuffering system and that penetrates into said analyte solution withinsaid chamber at the position of the first portion of the chemicalbuffering system in contact with the analyte solution, whereby compoundsthat are globally neutral at the pH or pH range defined by the firstportion of the chemical buffering system contacting the analyte solutionremain in said chamber upon potential application, whereas compoundsthat are charged at the pH or pH range defined by the first portion ofthe chemical buffering system contacting the analyte solution migrateand are extracted out of the chamber into said chemical buffering systemupon potential application.
 35. A kit according to claim 34, whereinsaid compounds that are globally neutral at the pH or pH range definedby the portion of the chemical buffering system contacting the analytesolution are recoverable in an ampholyte-free or buffer-free solution.36. A kit according to claim 34, further comprising specific chemicalsthat are necessary to mix with the analyte solution for theelectrophoretic separation and purification.
 37. A kit according toclaim 34, further comprising at least one of specific chemicals and aseparation system that is necessary to prepare the analyte solutionbefore the electrophoretic separation and purification.
 38. A kitaccording to claim 37, wherein said separation system is one of acut-off membrane and a desalting system.
 39. A kit according to claim34, wherein said chemical buffering system is selected from a groupconsisting of a gel comprising immobilised buffering molecules and abuffering fluid solidified in one of a polymer matrix, a fritted glass,a porous membrane, a filter and a combination thereof.
 40. A kitaccording to claim 34, wherein a length of said chamber is substantiallyperpendicular to a direction of the pH gradient of the chemicalbuffering system and to a direction of the electric field.